ABSTRACT
Replica molding (REM) is a powerful technique for fabricating anisotropic microparticles. Current REM methods rely on the use of gas-permeable molds for defect-free castings and facile particle recovery. However, they often encounter limitations on either technical accessibility or producible particle diversity. While the use of gas-impermeable molds presents a promising solution to these challenges, particle production within such molds necessitates addressing two critical issues: precursor loading and particle recovery. This study introduces a REM methodology specifically tailored to enable the production of anisotropic microparticles within gas-impermeable molds. To address the issue of precursor loading, our approach incorporates the air-through-precursor suction method, employing a degassed polydimethylsiloxane block to effectively eliminate air bubbles trapped in microwells. Additionally, fluorosilane pretreatment of the mold surface, along with the polyvinyl alcohol film formation, significantly enhances particle recovery up to 249-fold while ensuring particle homogeneity. This methodology demonstrates high adaptability to various gas-impermeable molds and curing techniques. The practical feasibility is illustrated through the successful production of functional composite microparticles that can be effectively utilized for oxygen sensing and self-assembly, challenging in conventional REM.
ABSTRACT
Multiplex detection of low-abundance protein biomarkers in biofluids can contribute to diverse biomedical fields such as early diagnosis and precision medicine. However, conventional techniques such as digital ELISA, microarray, and hydrogel-based assay still face limitations in terms of efficient protein detection due to issues with multiplexing capability, sensitivity, or complicated assay procedures. In this study, we present the degassed micromold-based particle isolation technique for highly sensitive and multiplex immunoassay with enzymatic signal amplification. Using degassing treatment of nanoporous polydimethylsiloxane (PDMS) micromold, the encoded particles are isolated in the mold within 5 min absorbing trapped air bubbles into the mold by air suction capability. Through 10 min of signal amplification in the isolated spaces by fluorogenic substrate and horseradish peroxidase labeled in the particle, the assay signal is amplified with one order of magnitude compared to that of the standard hydrogel-based assay. Using the signal amplification assay, vascular endothelial growth factor (VEGF) and chorionic gonadotropin beta (CG beta), the preeclampsia-related protein biomarkers, are quantitatively detected with a limit of detection (LoD) of 249 fg/mL and 476 fg/mL in phosphate buffer saline. The multiplex immunoassay is conducted to validate negligible non-specific detection signals and robust recovery rates in the multiplex assay. Finally, the VEGF and CG beta in real urine samples are simultaneously and quantitatively detected by the developed assay. Given the high sensitivity, multiplexing capability, and process simplicity, the presented particle isolation-based signal amplification assay holds significant potential in biomedical and proteomic fields.
Subject(s)
Biosensing Techniques , Limit of Detection , Vascular Endothelial Growth Factor A , Humans , Biosensing Techniques/methods , Immunoassay/methods , Vascular Endothelial Growth Factor A/urine , Vascular Endothelial Growth Factor A/isolation & purification , Vascular Endothelial Growth Factor A/analysis , Dimethylpolysiloxanes/chemistry , Chorionic Gonadotropin, beta Subunit, Human/urine , Chorionic Gonadotropin, beta Subunit, Human/isolation & purification , Chorionic Gonadotropin, beta Subunit, Human/blood , Chorionic Gonadotropin, beta Subunit, Human/analysis , Biomarkers/urine , Female , Pregnancy , Equipment DesignABSTRACT
The simultaneous quantification of multiple proteins is crucial for accurate medical diagnostics. A promising technology, the multiplex colorimetric immunoassay using encoded hydrogel microparticles, has garnered attention, due to its simplicity and multiplex capabilities. However, it encounters challenges related to its dynamic range, as it relies solely on the colorimetric signal analysis of encoded hydrogel microparticles at the specific time point (i.e., end-point analysis). This necessitates the precise determination of the optimal time point for the termination of the colorimetric reaction. In this study, we introduce real-time signal analysis to quantify proteins by observing the continuous colorimetric signal change within the encoded hydrogel microparticles. Real-time signal analysis measures the "slope", the rate of the colorimetric signal generation, by focusing on the kinetics of the accumulation of colorimetric products instead of the colorimetric signal that appears at the end point. By developing a deep learning-based automatic analysis program that automatically reads the code of the graphically encoded hydrogel microparticles and obtains the slope by continuously tracking the colorimetric signal, we achieved high accuracy and high throughput analysis. This technology has secured a dynamic range more than twice as wide as that of the conventional end-point signal analysis, simultaneously achieving a sensitivity that is 4-10 times higher. Finally, as a demonstration of application, we performed multiplex colorimetric immunoassays using real-time signal analysis covering a wide concentration range of protein targets associated with pre-eclampsia.
Subject(s)
Colorimetry , Hydrogels , Colorimetry/methods , Immunoassay/methods , Hydrogels/chemistry , Humans , Female , Pregnancy , Pre-Eclampsia/diagnosis , Deep LearningABSTRACT
The simultaneous genotyping of multiple single nucleotide polymorphisms (SNPs) in genomic DNA derived from organisms holds significant potential for applications such as precision medicine and food product authentication. However, conventional assay technologies including qPCR-based techniques, microarrays, and hydrogel-based assays face limitations in efficient multiplexing of SNPs, particularly for large-size DNA beyond kilobase scales, due to constraints in multiplex capability, specificity, or sensitivity. In this study, a hydrogel-based multiplex SNP genotyping platform specifically designed for genomic DNA is presented. This platform integrates the ligation detection reaction (LDR) and rolling circle amplification (RCA) techniques within a hydrogel-based multiplex sensing system, enabling adaptable and sensitive SNP genotyping for genomic DNA. To enhance the specificity of the assay, MutS protein and polyethylene glycol are introduced into the protocol, reducing the non-specific ligation and RCA reactions synergistically. With significant specificity improvement of over 10-fold, three types of SNPs within an artificially constructed â¼1000 bp double-stranded DNA (dsDNA) are successfully genotyped with double-digit picomolar sensitivity. Furthermore, the practical applicability of the developed process for the origin identification of raw materials is demonstrated by genotyping three types of SNPs within genomic DNA obtained from two closely related plant species, Korean ginseng (Panax ginseng) and American ginseng (Panax quinquefolius), containing ca. 3.5 gigabase genome size. Of notable significance, this study marks the premiere achievement in PCR-free multiplex genotyping of SNPs in genomic DNA using a single fluorophore.
ABSTRACT
Graphically encoded hydrogel microparticle (HMP)-based bioassay is a diagnostic tool characterized by exceptional multiplex detectability and robust sensitivity and specificity. Specifically, deep learning enables highly fast and accurate analyses of HMPs with diverse graphical codes. However, previous related studies have found the use of plain particles as data to be disadvantageous for accurate analyses of HMPs loaded with functional nanomaterials. Furthermore, the manual data annotation method used in existing approaches is highly labor-intensive and time-consuming. In this study, we present an efficient deep-learning-based analysis of encoded HMPs with diverse graphical codes and functional nanomaterials, utilizing the auto-annotation and synthetic data mixing methods for model training. The auto-annotation enhanced the throughput of dataset preparation up to 0.11 s/image. Using synthetic data mixing, a mean average precision of 0.88 was achieved in the analysis of encoded HMPs with magnetic nanoparticles, representing an approximately twofold improvement over the standard method. To evaluate the practical applicability of the proposed automatic analysis strategy, a single-image analysis was performed after the triplex immunoassay for the preeclampsia-related protein biomarkers. Finally, we accomplished a processing throughput of 0.353 s per sample for analyzing the result image.
Subject(s)
Deep Learning , Hydrogels , Image Processing, Computer-Assisted/methods , Biomarkers , Immunoassay/methodsABSTRACT
Controlled and targeted delivery of growth factors to biological environments is important for tissue regeneration. Polylactic acid (PLA) hydrogel microparticles are attractive carriers for the delivery of therapeutic cargoes based on their superior biocompatibility and biodegradability, uniform encapsulation of cargoes, and non-requirement of organic solvents during particle synthesis. In this study, we newly present controlled growth factor delivery utilizing PLA-based hydrogel microcarriers synthesized via degassed micromolding lithography (DML). Based on the direct gelation procedure from the single-phase aqueous precursor in DML, bovine serum albumin, a model protein of growth factor, and fibroblast growth factor were encapsulated into microparticles with uniform distribution. In addition, by tuning the monomer concentration and adding a hydrolytically stable crosslinker, the release of encapsulated cargoes was efficiently controlled and extended to 2 weeks. Finally, we demonstrated the biological activity of encapsulated FGF-2 in PLA-based microparticles using a fibroblast proliferation assay.
Subject(s)
Hydrogels , Polyesters , Intercellular Signaling Peptides and Proteins , Solvents , Particle SizeABSTRACT
Several multiplex nucleic acid assay platforms have been developed in response to the increasing importance of nucleic acid analysis, but these assays should be optimized as per the requirements of point-of-care for clinical diagnosis. To achieve rapid and accurate detection, involving a simple procedure, we propose a new concept in the field of nucleic acid multiplex assay platforms using hydrogel microparticles, called barcode receptor-encoded particles (BREPs). The BREP assay detects multiple targets in a single reaction with a single fluorophore by analyzing graphically encoded hydrogel particles. By introducing sets of artificially synthesized barcode receptor and barcode probes, the BREP assay is easily applicable in multiplexing any genetic target; sets of barcode receptors and barcode probes should be designed delicately for universal application. The performance of the BREP assay was successfully verified in a multiplex assay for the identification of different malaria species with high sensitivity, wide dynamic range, fast detection time, and multiplexibility.
ABSTRACT
Replica molding is widely used to reproduce the surface microstructures that provide living organisms with distinct and useful functions. However, the existing methods are limited by the low resolution resulting from the air trapped in the structures during precursor solution loading. This study investigated replica molding with an air-through-precursor suction (APS) process, which used a degassed polydimethylsiloxane substrate to remove the trapped air through the precursor solution. The liquid loading times are characterized using a model template, and air suction that is up to 36 times faster can be achieved using the APS process relative to a conventional method. Using APS replica molding, biocompatible replicates from human fingerprints and gecko skin are fabricated using only a 3 min precursor solution loading step. Owing to the enhanced and reproducible resolution from APS replica molding, for the first time, the structural changes in the foot of a living gecko at the microscale can be observed when standing on a horizontal or vertical surface.
Subject(s)
Suction , HumansABSTRACT
Hydrogel microparticle-based nucleic acid assays are an attractive detection platform based on their multiplexing capabilities with high sensitivity and specificity. A particular area of interest is single-nucleotide polymorphism (SNP) sensing, where multiple SNPs should be identified in a highly reliable yet economical manner. However, hydrogel microparticles leveraging probe-target hybridization as a key mechanism are hampered by small duplex stability differences arising from single base-pair mismatch. We have developed encoded hydrogel microparticles with DNA probes tailored for multiplex SNP detection. Within the DNA probes, we adopt a widely used base analog (5-nitroindole) so that it substitutes one of the base sequences among DNA probes. The effects of the modification of the probes' structure on SNP sensing has been tested from multiple perspectives, such as specificity, sensitivity, and available assay temperatures at a given ionic strength. We have validated that our hydrogel microparticles exhibit much higher specificity for a single base-pair mismatch with minimal reduction in sensitivity. Our particles can also detect multiple SNPs located in different target strands, which is a significant challenge for conventional particles.
Subject(s)
Hydrogels , Polymorphism, Single Nucleotide , DNA , DNA Probes/chemistry , DNA Probes/genetics , Hydrogels/chemistry , Nucleic Acid HybridizationABSTRACT
Encoded hydrogel microparticles mounting DNA probes are powerful tools for high-performance microRNA (miRNA) detection in terms of sensitivity, specificity, and multiplex detection capability. However, several particle rinsing steps in the assay procedure present challenges for rapid and efficient detection. To overcome this limitation, we encapsulated dense magnetic nanoparticles to reduce the rinsing steps and duration via magnetic separation. A large number of magnetic nanoparticles were encapsulated into hydrogel microparticles based on a discontinuous dewetting technique combined with degassed micromolding lithography. In addition, we attached DNA probes targeting three types of miRNAs related to preeclampsia to magnetically encoded hydrogel microparticles by post-synthesis conjugation and achieved sensitivity comparable to that of conventional nonmagnetic encoded hydrogel microparticles. To demonstrate the multiplex capability of magnetically encoded hydrogel microparticles while maintaining the advantages of the simplified rinsing process when addressing multiple samples, we conducted a triplex detection of preeclampsia-related miRNAs. In conclusion, the introduction of magnetically encoded hydrogel microparticles not only allowed efficient miRNA detection but also provided comparable sensitivity and multiplexed detectability to conventional nonmagnetic encoded hydrogel microparticles.
ABSTRACT
microRNAs (miRNAs) have attracted much attention as potential biomarkers for the diagnosis of various fatal diseases. With increasing interest in miRNA detection at practical sites, colorimetric bead-based assays have garnered much attention, because these allow for simple analysis with cheap and portable devices. Among them, the encoded hydrogel microparticle-based colorimetric miRNA assay is considered as one of the promising techniques, due to its strengths, such as large multiplex capacity, acceptable sensitivity, and simple analysis. However, it still imposes a limitation in terms of the assay time, particularly the colorimetric reaction time, which is too long, making the practical application of the assay difficult and undermining its detection accuracy. In this work, we present a rapid colorimetric assay based on encoded hydrogel microparticles, which exhibits a significant decrease in the colorimetric reaction time due to two factors: (1) an increase in the number of enzymes bound to hydrogel microparticles via a post-synthesis functionalization method, and (2) an elevation in the enzyme reaction temperature during colorimetric labeling. We obtained a comparable sensitivity of the colorimetric assay with three different miRNA targets, even with a shortened colorimetric reaction time. Furthermore, we validated that our colorimetric detection method is suitable for multiplex miRNA detection, owing to its low cross-reactivity.
Subject(s)
Colorimetry , MicroRNAs , Biomarkers , Hydrogels , MicroRNAs/geneticsABSTRACT
Although urinary exosomal microRNAs (miRNAs) have recently emerged as potential biomarkers, clinical applications are still limited due to their low concentration in small volumes of clinical samples. Therefore, the development of a non-invasive, specific diagnostic tool, along with profiling exosomal miRNA markers from urine, remains a significant challenge. Here, we present hydrogel-based hybridization chain reaction (HCR) for multiplex signal amplification to detect urinary exosomal miRNAs from human clinical samples. We succeeded in identifying small amounts (~amol) of exosomal miRNAs from 600 µL of urine with up to ~35-fold amplification and enhanced detection limits by over an order of magnitude for two miRNA biomarker candidates, hsa-miR-6090 and hsa-miR-3665. Furthermore, we proposed ratiometric analysis without requiring normalization to a reference miRNA and validated the clinical diagnostic potential toward differentiating prostate cancer patients from healthy controls. Our hydrogel-based HCR could serve as a new diagnostic platform for a non-invasive liquid biopsy before burdensome tissue biopsy of various diseases, including prostate cancer screening, complementing the PSA test.
Subject(s)
Biosensing Techniques , Exosomes , MicroRNAs , Prostatic Neoplasms , Biomarkers, Tumor/genetics , Early Detection of Cancer , Exosomes/genetics , Humans , Hydrogels , Male , MicroRNAs/genetics , Prostate-Specific Antigen , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/geneticsABSTRACT
Discontinuous dewetting (DD) is an attractive technique that enables the production of large liquid arrays in microwells and is applicable to the synthesis of anisotropic microparticles with complex morphologies. However, such loading of liquids into microwells presents a significant challenge, as the liquids used in this technique should exhibit low mold surface wettability. This study introduces DD in a degassed mold (DM), a simple yet powerful technique that achieves uniform loading of microparticle precursors into large microwell arrays within 1 min. Using this technique, hydrogel microparticles are produced by different polymerization mechanisms with various shapes and sizes, ranging from a few micrometers to hundreds of micrometers. Hydrophobic oil microparticles are produced by the simple plasma treatment of the DM, and agarose microparticles encapsulating bovine serum albumin (in a well-dispersed state) are produced by submerging the DM in fluorinated oil. To demonstrate additional functionality of microparticles using this technique, high concentrations of magnetic nanoparticles are loaded into microparticles for particle-based immunoassays performed in a microwell plate, and the immunoassay performance is comparable to that of ELISA.
Subject(s)
Microspheres , Polymers/chemistry , Humans , Hydrogels/chemistry , Immunoassay/methods , Magnetite Nanoparticles/chemistry , Placenta Growth Factor/analysis , WettabilityABSTRACT
Magnetic hydrogels have been commonly used in biomedical applications. As magnetite nanoparticles (MNPs) exhibit peroxidase enzyme-like activity, magnetic hydrogels have been actively used as signal transducers for biomedical assays. Droplet microfluidics, which uses photoinitiated polymerization, is a preferred method for the synthesis of magnetic hydrogels. However, light absorption by MNPs makes it difficult to obtain fully polymerized and homogeneous magnetic hydrogels through photoinitiated polymerization. Several methods have been reported to address this issue, but few studies have focused on investigating the light absorption properties of photoinitiators. In this study, we developed a simple method for the synthesis of poly(ethylene glycol) (PEG)-based uniform magnetic hydrogels that exploits the high ultraviolet absorption of a photoinitiator. Additionally, we investigated this effect on shape deformation and structural uniformity of the synthesized magnetic hydrogels. Two different photoinitiators, Darocur 1173 and lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP), with significantly different UV absorption properties were evaluated based on the synthesis of magnetic hydrogels. The magnetic characteristics of the PEG-stabilized MNPs in hydrogels were investigated with a vibrating sample magnetometer. Finally, the colorimetric detection of hydrogen peroxide and glucose was conducted based on the enzyme-like property of MNPs and repeated several times to observe the catalytic activity of the magnetic hydrogels.
ABSTRACT
Despite a growing demand for more accessible diagnostic technologies, current methods struggle to simultaneously detect multiple analytes with acceptable sensitivity and portability. Colorimetric assays have been widely used due to their simplicity of signal readout, but the lack of multiplexibility has been a perpetual constraint. Meanwhile, particle-based assays offer multiplex detection by assigning an identity code to each analyte, but they often require lab-based equipment unsuitable for portable diagnostics. Here, by merging the two approaches, this paper reports a colorimetric multiplex immunoassay based on hydrogel microparticles that achieves the best of both worlds. The low-cost portable multiplex assay demonstrates sensitivities as high as and dynamic ranges greater than the lab-based enzyme-linked immunosorbent assay (ELISA). These critical advances are made possible by local precipitation and amplification of insoluble colour dyes inside the hydrogel networks. For the first time, enzymatic accumulation of colour dyes in hydrogel particles is reported and the kinetics of colour development is characterized in this work. By taking advantage of the colour signals in the visible spectrum, the hydrogel microparticles were imaged and analysed using low-cost portable devices. The colorimetric multiplex immunoassay was used to successfully detect three target biomarkers of preeclampsia and validated clinically using healthy and patient-derived plasma samples.
Subject(s)
Colorimetry , Hydrogels , Biomarkers , Enzyme-Linked Immunosorbent Assay , Humans , ImmunoassayABSTRACT
Due to the growing interest in multiplex protein detection, encoded hydrogel microparticles have received attention as a possible path to high performance multiplex immunoassays through a combination of high multiplexing capability and enhanced binding kinetics. However, their practical operation in real complex samples is still limited because polyethylene glycol, which is the main component of hydrogel particles, suffers from oxidative damage and relatively high fouling properties in biochemical solutions. Here, we introduce poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-based encoded hydrogel microparticles to perform fouling-resistant multiplex immunoassays, where the anti-fouling characteristics are attributed to the zwitterionic PMPC. By applying a newly developed molding lithography technique, viscous PMPCs with low reactivity were successfully incorporated into the hydrogel network while maintaining uniformity and rigidity for use in multiplex immunoassays. Non-specific protein adsorption on the PMPC particles was reduced by about 37.5% compared to that of conventional PEG particles, which leads to better assay sensitivity. We also validate the multiplex capability of the PMPC particles by performing multiplex detection of two target proteins. Furthermore, we verify that the PMPC particles have a 70% enhancement in anti-fouling characteristics compared to PEG particles in human platelet-rich plasma, potentiating a practical immunoassay platform for clinical diagnosis.
Subject(s)
Hydrogels , Phosphorylcholine , Adsorption , Humans , Immunoassay , Polyethylene GlycolsABSTRACT
Encoded hydrogel microparticles synthesized via flow lithography have drawn attention for multiplex biomarker detection due to their high multiplex capability and solution-like hybridization kinetics. However, the current methods for preparing particles cannot achieve a flexible, rapid probe-set modification, which is necessary for the production of various combinations of target panels in clinical diagnosis. In order to accomplish the unmet needs, streptavidin was incorporated into the encoded hydrogel microparticles to take advantage of the rapid streptavidin-biotin interactions that can be used in probe-set modification. However, the existing methods suffer from low efficiency of streptavidin conjugation, cause undesirable deformation of particles, and impair the assay capability. Here, we present a simple and powerful method to conjugate streptavidin to the encoded hydrogel microparticles for better assay performance and rapid probe-set modification. Streptavidin was directly conjugated to the encoded hydrogel microparticles using the aza-Michael addition click reaction, which can proceed in mild, aqueous condition without catalysts. A highly flexible and sensitive assay was developed to quantify DNA and proteins using streptavidin-conjugated encoded hydrogel microparticles. We also validated the potential applications of our particles conducting multiplex detection of cancer-related miRNAs.
ABSTRACT
Flow lithography (FL), a versatile technique used to synthesize anisotropic multifunctional microparticles, has attracted substantial interest, given that the resulting particles with complex geometries and multilayered biochemical functionalities can be used in a wide variety of applications. However, after this process, there are double bonds remaining from the cross-linkable groups of monomers. The unreacted cross-linkable groups can affect the particles' biochemical properties. Here, we verify that the microparticles produced by FL contain a significant number of unreacted acrylate double bonds (UADBs), which could cause irreversible biochemical changes in the particle and pernicious effects to biological systems. We also confirm that the particles contain a considerable number of UADBs, regardless of the various synthetic (lithographic) conditions that can be used in a typical FL process. We present an effective way to eliminate a substantial amount of UADBs after synthesis by linking biochemically inert poly(ethylene glycol) based on click chemistry. We verify that eliminating UADBs by using this click chemistry approach can efficiently resolve problems, such as the occurrence of random reactions and the cytotoxicity of UADBs.
ABSTRACT
Technologies for the detection and isolation of circulating tumor cells (CTCs) are essential in liquid biopsy, a minimally invasive technique for early diagnosis and medical intervention in cancer patients. A promising method for CTC capture, using an affinity-based approach, is the use of functionalized hydrogel microparticles (MP), which have the advantages of water-like reactivity, biologically compatible materials, and synergy with various analysis platforms. In this paper, we demonstrate the feasibility of CTC capture by hydrogel particles synthesized using a novel method called degassed mold lithography (DML). This technique increases the porosity and functionality of the MPs for effective conjugation with antibodies. Qualitative fluorescence analysis demonstrates that DML produces superior uniformity, integrity, and functionality of the MPs, as compared to conventional stop flow lithography (SFL). Analysis of the fluorescence intensity from porosity-controlled MPs by each reaction step of antibody conjugation elucidates that more antibodies are loaded when the particles are more porous. The feasibility of selective cell capture is demonstrated using breast cancer cell lines. In conclusion, using DML for the synthesis of porous MPs offers a powerful method for improving the cell affinity of the antibody-conjugated MPs.
ABSTRACT
Replica molding techniques, which are used to synthesize microparticles inside anisotropic micromolds, have been developed to enable the mass production of hydrogel particles. However, these techniques are limited in their ability to synthesize only a narrow range of particle compositions and shapes because of the difficulty in loading precursors into the micromolds as well as the low particle homogeneity due to the uneven evaporation of the precursors. Herein, we describe a simple yet powerful technique, called degassed micromolding lithography, which can load precursors within 1 min regardless of the wettability. This technique is based on the gas-solubility of a degassed micromold that acts as a suction pump to completely fill the mold by drawing precursor liquids in. The semi-closed system within the micromold prevents the uneven evaporation of the precursor, which is essential for the production of homogeneous particles. Furthermore, controlled uniformity of the hydrogel microparticles (C.V. < 2%) can be achieved by engineering the design of the micromold array.